Nonalcoholic fatty liver disease (NAFLD) occurs in patients without significant alcohol consumption and represents a clinico-histopathological entity with histological features that resemble alcohol-induced liver injury. We have used conditional gene targeting in hepatocytes to inactivate the pVHL-E3-ubiquitin ligase, which targets Hypoxia-Inducible-Factor (HIF) for degradation under normoxia. We have discovered that increased HIF activity alters fatty acid metabolism and results in the development of non-alcoholic fatty liver disease in mice. Hypoxia-Inducible Factor-1 and -2 (HIF-1 and HIF-2) are heterodimeric basic-loop-helix transcription factors and are key mediators of cellular adaptation to diminished oxygen supply. Our findings implicate HIF signaling, in particular signaling through HIF-2, in the development of fatty liver disease. In this grant application we propose in vivo and in vitro studies that make use of conditional gene targeting technology and transgenesis to investigate the role of HIF signaling in the regulation of fatty acid uptake, synthesis, beta-oxidation and secretion. Additional in vitro studies are proposed that specifically focus on selected HIF target genes to study their role in lipid metabolism under hypoxia. In Aim 1 we carry out functional studies in VHL mutant mice, in Aim 2 we investigate the role of HIF activation early in the development of steatosis following acute inactivation of pVHL in the adult, and Aims 3 and 4 investigate the role of HIF-2 in a wild type genetic background with a focus on HIF-2 target genes relevant for the development of steatosis. The proposed studies are not only important for our understanding of basic HIF functions in lipid metabolism, but more importantly have direct clinical relevance. We provide a direct molecular link between hypoxic injury and fatty liver development and establish a novel role for the HIF pathway in the pathogenesis of NAFLD.